Active fragments from pro- and antiapoptotic BCL-2 proteins have distinct membrane behavior reflecting their functional divergence

Abstract

Background: The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction.

Methodology/principal findings: Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death.

Conclusion/significance: BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains.

Figure 1. Aligned aminoacid sequences of the different BCL-2 family proteins investigated in this study.

The conserved BH1-3 domains are indicated. BID shows similarity only in the BH3 region. The sequence and some general properties of the peptides analyzed are described in Table 1.

Figure 2. Surface pressure-time isotherms for BAX-α5S with (bold line) or without (light line) POPC/DOPE/CL lipids.

The peptide was added to the subphase at a 0.2 µM concentration and the increment of π after addition of the peptide was complete in ∼1 h. Δπ was taken to be the difference between the initial surface pressure (π_i = 5 mN/m) and the value (π_max) observed after the penetration of the peptide into the lipid monolayer. The initial velocity of surface pressure increase (Vi) was calculated as the slope of the curve (Δπ/Δt) at the time of peptide addition. When the peptide was injected into the subphase in the absence of a lipid monolayer, the system was allowed to stabilize for 10 min and a compression at π_i = 5 mN/m (the same initial surface pressure of the lipid monolayer) was applied to ensure as similar as possible interfacial conditions.

Figure 3. Plot of surface pressure increase versus initial surface pressure.

Maximal surface pressure increase (Δπ) induced by injection of the Bax-α5S peptide underneath a POPC/DOPE/CL monolayer, as a function of various initial surface pressures (π_i). The exclusion pressure (π_ex) was determined from the abscissa intercept.

Figure 4. Plot of surface pressure versus mean molecular area (MMA).

The grey lines depict the compression-decompression isotherm obtained without peptide (POPC/DOPE/CL monolayer only). The gas-like phase is present near the onset of pressure at the surface of the interface. The monolayer then changes to a liquid-expanded phase. In the presence of BAX-α5S injected at a concentration of 0.2 µM, the isotherms show a shift toward larger area (black lines), indicating peptide incorporation into the monolayer. The dashed lines were obtained after increasing the surface pressure to π>π_ex and indicate that the peptide was in part squeezed out from the monolayer, hysteresis being probably due to peptide ejection.

Figure 5. Changes in surface pressure after peptide injection.

Peptides were injected underneath POPC/DOPE or POPC/DOPE/CL monolayers at constant area. A. Final increase of surface pressure π_max (mN/m) obtained for the different peptides. The dashed line denotes the threshold (Δπ = 4.8 mN/m) for significant surface pressure variation as determined using BAX-α5S without lipids (see text). B. Initial velocity of surface pressure increase (V_i = Δπ/s).

Figure 6. Topographic structure of the monolayers visualized by Brewster angle microscopy.

BAM images were recorded at π_i ( = 5 mN/m) and once the plateau surface pressure (π_max) was attained. Scale bars are included. A. BAM microphotographs for the first helix (BAX-α5S, BCLX-α5S, BID-α6) of the ‘pore domain’ of BAX, BCL-xL and BID. B. BAM images in presence of the second helix (BAX-α6, BCLX-α6, BID-α7) of the ‘pore domain’. Zoomed cutouts (low panels) are depicted for BAX-α5s (in A) and BAX-α6 (in B). C. BAM images in presence of the BH3 peptides. D. BAM images for BCLX-α5L and BAX-α5L, M and MS. E. BAM pictures for BAX-α5M in pure DOPE monolayers at the indicated time after peptide injection.

Figure 7. Cytochrome c release assays.

Peptides were incubated with isolated mitochondria for the indicated times (min) and the release of cytochrome c was monitored by Western blotting (IB). MitoHSP70 was used as an equal-loading control for the pellet fraction. Control lanes indicate that in the preparation the MOM is intact and cytochrome c is retained within the intermembrane space. A. Cytochrome c release assays for the first (BAX-α5S, BCLX-α5S, BID-α6) and second helices (BAX-α6, BCLX-α6, BID-α7) of the ‘pore domain’ of BAX, BCL-xL and BID using mitochondria isolated from HEK293T cells. B. Cytochrome c release assays for BAX-α5M and BAX-α5MS using mitochondria isolated from MEF and MEK BAX/BAK -/- double knock-out cells (MEF DKO). C. Cytochrome c release assays for the BH3 peptides.